Does Plant Cell Death Does Plant Cell Death Induced by Ptr ToxA Induced by Ptr ToxA RequireRequire Toxin Entry? Toxin Entry?

Sara M. HamiltonSara M. Hamilton

Viola A. ManningViola A. Manning

Dr. Lynda M. CiuffettiDr. Lynda M. Ciuffetti

Department of Botany and Plant PathologyDepartment of Botany and Plant Pathology

PyrenophoraPyrenophora triticitritici--repentisrepentis

Filamentous fungus-ascomyceteFilamentous fungus-ascomycete Plant pathogen causing the disease Plant pathogen causing the disease tantan spotspot of of

sensitive wheat speciessensitive wheat species Crop losses estimated up to 50% in susceptible Crop losses estimated up to 50% in susceptible

varieties worldwidevarieties worldwide

Races of Races of Pyrenophora tritici-repentisPyrenophora tritici-repentis

N = causes necrosisN = causes necrosis C = causes chlorosisC = causes chlorosis R = resistant to pathogenR = resistant to pathogen

Race 1Race 2

Race 3

Race 4

Race 5

SalamouniGlenlea

N (ToxA)

N (ToxA)

R

R

R

R

R

R

R

R

Katepwa

N (ToxA)

N (ToxA)

R

R

C (ToxB)

6B365

C (ToxC)

R

C (ToxC)

R

R

6B662

R

R

R

R

C (ToxB)

Ptr ToxAPtr ToxA

First host selective toxin (HST) isolated from First host selective toxin (HST) isolated from P. P. tritici-repentistritici-repentis

First proteinaceous HSTFirst proteinaceous HST

Encoded by a single gene, the Encoded by a single gene, the ToxAToxA gene gene

Ptr ToxAPtr ToxA

Causes necrosis on sensitive wheat cultivarsCauses necrosis on sensitive wheat cultivars Does not require pathogen to cause disease Does not require pathogen to cause disease

symptomssymptoms

Sensitive

Insensitive

We want to know:We want to know:

1. What part of the ToxA protein is 1. What part of the ToxA protein is necessary for disease symptoms?necessary for disease symptoms?

2. Where does the protein exert activity 2. Where does the protein exert activity (i.e. where is the site-of-action)? (i.e. where is the site-of-action)?

Question #1Question #1

What part of the ToxA protein is What part of the ToxA protein is necessary for disease?necessary for disease?

Conserved ToxA Motifs Conserved ToxA Motifs

““RGD” cell attachment site RGD” cell attachment site RGD sites mediate interaction of cell matrix RGD sites mediate interaction of cell matrix

proteins with a family of membrane-bound proteins with a family of membrane-bound receptors called integrins.receptors called integrins.

Casein kinase II (CKII) and Protein kinase C Casein kinase II (CKII) and Protein kinase C (PKC) phosphorylation sites(PKC) phosphorylation sites

QGSCMSITINPSRPSVNNIGQVDIDSVILGRPGAIGSWELNNFITIGLNRVNADTVRVNIRNTGRTNRLIITQWDNTVTRGDVYELFGDYALIQGRGSFCLNIRSDTGRENWRMQLEN

ToxA Protein SequenceToxA Protein Sequence

Both the RGD and casein kinase II phosphorylation motifs are required for ToxA activity

Question #2Question #2

Where does the protein exert activity Where does the protein exert activity (i.e. where is the site-of-action)? (i.e. where is the site-of-action)?

ToxA LocalizationToxA Localization

ToxA is imported into mesophyll cells of ToxA is imported into mesophyll cells of sensitive wheat genotypes and localizes to the sensitive wheat genotypes and localizes to the chloroplasts of these cells.chloroplasts of these cells.

ToxA localization can be visualized ToxA localization can be visualized in vivoin vivo by by treatment of wheat with a green flourescent treatment of wheat with a green flourescent protein (GFP) fused to ToxA (GFP-ToxA).protein (GFP) fused to ToxA (GFP-ToxA).

GFP-ToxA: GFP-ToxA: Localization to ChloroplastsLocalization to Chloroplasts

Sensitive Insensitive

ToxA

GFP-ToxA

HypothesisHypothesis

ToxA entry into mesophyll cells is ToxA entry into mesophyll cells is required to cause cell death.required to cause cell death.

Current StudyCurrent Study

Produce GFP-ToxA proteins harboring Produce GFP-ToxA proteins harboring mutations and determine their localization mutations and determine their localization inin plantaplanta

Mutations include amino acids in the RGD cell Mutations include amino acids in the RGD cell attachment site and phosphorylation motifs.attachment site and phosphorylation motifs.

GFP-ToxA: GFP-ToxA: Construction of Fusion Protein VectorConstruction of Fusion Protein Vector

Green Fluorescent Protein

Ptr ToxA

GFP-ToxA MutantsGFP-ToxA Mutants

Mutagenize parent GFP-ToxA plasmid:Mutagenize parent GFP-ToxA plasmid:

Site-directed mutagenesisSite-directed mutagenesis Subcloning from previously mutagenized ToxA Subcloning from previously mutagenized ToxA

constructsconstructs

PCR site-directed mutagenesis proved to be PCR site-directed mutagenesis proved to be more efficient than subcloning.more efficient than subcloning.

Mutationto Alanine

Method of Mutagenesis Motif Mutagenized

t63 subcloned PKC

t66 site-directed PKC

n76 site-directed *Essential A.A.

t77 subcloned *Essential A.A.

v78 site-directed *Essential A.A.

t79 site-directed CKII

r80 site-directed RGD

g81 site-directed RGD

d82 subcloned RGD

v83 site-directed *Essential A.A.

e85 site-directed *Essential A.A.

Mutations of GFP-ToxAMutations of GFP-ToxA

* Essential amino acids surround the RGD motif

Expression of GFP-ToxAExpression of GFP-ToxA

Transformation of Transformation of E. coliE. coli with vector with vector

Expression of GFP-ToxA in Expression of GFP-ToxA in E. coliE. coli

Purification of GFP-ToxAPurification of GFP-ToxA

Protein PurificationProtein Purification

kDa

24

72

33

40

55

pCVM77 fusion protein gelpCVM77 fusion protein gel

To Be Completed:To Be Completed:

Infiltration of GFP-ToxA mutant proteins into Infiltration of GFP-ToxA mutant proteins into sensitive/insensitive wheat leaves:sensitive/insensitive wheat leaves:

Assay activityAssay activity

Determine localization via fluorescent Determine localization via fluorescent microscopymicroscopy

Dissecting the ToxA PathwayDissecting the ToxA Pathway

This information will allow us to determine if the This information will allow us to determine if the mutant proteins synthesized will:mutant proteins synthesized will:

Cross the cell wallCross the cell wall

Cross the plasma membraneCross the plasma membrane

Localize to an organelle (ex. chloroplast)Localize to an organelle (ex. chloroplast)

AcknowledgementsAcknowledgements

Howard Hughes Medical Howard Hughes Medical InstitutionInstitution

Ernest and Pauline JaworskiErnest and Pauline Jaworski USDAUSDA

Dr. Kevin AhernDr. Kevin Ahern

Dr. Lynda M. CiuffettiDr. Lynda M. Ciuffetti Viola A. ManningViola A. Manning

Dr. Pat MartinezDr. Pat MartinezDr. Iovanna PandelovaDr. Iovanna PandelovaKristin SkinnerKristin SkinnerRachael AndrieRachael AndrieRebecca Tippner-Rebecca Tippner- HedgesHedgesAlex BabininAlex Babinin